The phosphate signal transduction (PHO) pathway, which regulates genes in response to phosphate starvation, is well defined in Saccharomyces cerevisiae. We asked whether the PHO pathway was the same in the distantly related fission yeast Schizosaccharomyces pombe. We screened a deletion collection for mutants aberrant in phosphatase activity, which is primarily a consequence of pho1 ؉ transcription. We identified a novel zinc finger-containing protein (encoded by spbc27b12.11c ؉ ), which we have named pho7 ؉ , that is essential for pho1؉ transcriptional induction during phosphate starvation. Few of the S. cerevisiae genes involved in the PHO pathway appear to be involved in the regulation of the phosphate starvation response in S. pombe. Only the most upstream genes in the PHO pathway in S. cerevisiae (ADO1, DDP1, and PPN1) share a similar role in both yeasts. Because ADO1 and DDP1 regulate ATP and IP 7 levels, we hypothesize that the ancestor of these yeasts must have sensed similar metabolites in response to phosphate starvation but have evolved distinct mechanisms in parallel to sense these metabolites and induce phosphate starvation genes.The cellular homeostasis of inorganic phosphate is required for optimal growth and efficient metabolism. The response of the model organism Saccharomyces cerevisiae to extracellular phosphate starvation is well characterized and mediated by the phosphate signal transduction (PHO) pathway (20,24). To determine whether the PHO pathway is conserved in other Ascomycota fungal species, we screened for PHO pathway mutants in the evolutionarily distantly related Schizosaccharomyces pombe, which last shared a common ancestor with S. cerevisiae more than 1 billion years ago (7).The PHO pathway in S. cerevisiae often is defined by the regulation of PHO5, which encodes a phosphate starvationregulated acid phosphatase (17,20). PHO5 is highly induced during phosphate starvation. ScPho5 activity is detected using a diazo-coupling assay with 1-napthylphosphate (9). Numerous studies have determined that PHO5 transcription is regulated by the specific transcription factors Pho4 and Pho2 and by more general chromatin remodeling complexes, such as SWI/ SNF, SAGA, and INO80 (1, 16, 31). Pho4 localization and activity is regulated by a cyclin/cyclin-dependent kinase complex (Pho81/Pho80/Pho85) (11,12,25). During high extracellular phosphate conditions, the kinase complex is active and phosphorylates Pho4, leading to nuclear exclusion and little transcription of PHO5 (10, 15). During low extracellular phosphate conditions, Pho81 inhibits the kinase complex through a noncovalent interaction with IP 7 (inositol heptakisphosphate) (18,19). Certain isomers of IP 7 increase in abundance in response to phosphate starvation, although how extracellular phosphate concentration leads to these increases is unclear. However, Vip1 is required to phosphorylate IP 6 to form 4-PP-IP 5 or 6-PP-IP 5 , and Ddp1 is required for dephosphorylation back to IP 6 (19). Increases in IP 7 during extracellular phosphate st...

Background Recent evidence suggests that storage induced alterations of the red blood cell (RBC) are associated with adverse consequences in susceptible hosts. As RBCs have been shown to form Advanced Glycation Endproducts (AGEs) following increased oxidative stress and under pathologic conditions, we examined whether stored RBCs undergo modification with the specific AGE, N-(Carboxymethyl)lysine (Nε-CML) during standard blood banking conditions. Study Design and Methods Purified, fresh RBCs from volunteers were compared to stored RBCs (d 35–42 old) obtained from the Blood Bank. Nε-CML formation was quantified using a competitive enzyme-linked immunosorbent assay. The receptor for advanced glycation end-products (RAGE) was detected in human pulmonary microvascular endothelial cells by real-time PCR, western blotting, and flow cytometry. Intracellular reactive oxygen species (ROS) generation was measured by the use of 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester based assays. Results Stored RBCs showed increased surface Nε-CML formation when compared with fresh RBCs. Human Pulmonary Microvascular Endothelial Cells (HMVEC-L) showed detectable surface RAGE expression constitutively. When compared to fresh RBCs, stored RBCs triggered increased intracellular ROS generation in both Human Umbilical Vein Endothelial Cells (HUVEC) and Human Pulmonary Microvascular Endothelial Cells (HMVEC-L). RBC-induced endothelial ROS generation was attenuated in the presence of soluble RAGE (sRAGE) or RAGE blocking antibody. Conclusion The formation of the AGE Nε-CML on the surface of stored RBCs is one functional consequence of the storage lesion. AGE-RAGE interactions may be one mechanism by which transfused RBCs cause endothelial cell damage.

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